Novel Anti-Gprc5d Antibodies, Bispecific Antigen Binding Molecules That Bind Gprc5d and Cd3, and Uses Thereof

The present disclosure generally relates to novel anti-GPRC5D antibodies and antibody fragments thereof, bispecific antigen binding molecules that bind GPRC5D and CD3, and uses of the same.

G-protein coupled receptor, class C, group 5, member D (GPRC5D) is an orphan, atypical, class C G-protein coupled receptor (GPCR) first identified in 2001 (Brauner-Osborne et al.,1518(3):237-248, 2001). GPRC5D is a specific surface protein expressed by plasma cells in multiple myeloma, and might be a relevant target on plasma cells in autoimmunity as well. It has been reported that GPRC5D is associated with prognosis and tumor load in multiple myeloma patients (Atamaniuk, J., et al.,42(9): 953-960, 2012; and Cohen, Y., et al.,18(6): 348-351, 2013). Various anti-GPRC5D antibodies have been described in the field, for example, in WO2018017786A2 and WO2021018925A1.

The CD3 (cluster of differentiation 3) T-cell co-receptor is a protein complex and is composed of four distinct chains, a CD3gamma chain, a CD3delta chain, and two CD3epsilon chains. These chains associate with a molecule known as the T-cell receptor (TCR) and the zeta-chain to generate activation signal in T lymphocytes. The TCR, zeta-chain, and CD3 molecules together form the TCR-CD3 complex, in which TCR as a subunit recognizes and binds to antigen, and CD3 as a subunit transfers and conveys the antigen-stimulation to signaling pathway, and ultimately regulates T-cell activity. The CD3 protein is virtually present in all T cells. A more recent application of CD3 antibodies is in the form of bispecific antibodies, binding CD3 on the one hand and a tumor cell antigen on the other hand. The simultaneous binding of such an antibody to both of its targets will force a temporary interaction between target cell and T cell, causing activation of any cytotoxic T cell and subsequent lysis of the target cell.

Needs remain for additional drugs to treat cancer, particularly multiple myeloma.

Throughout the present disclosure, the articles “a”, “an”, and “the” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an antibody” means one antibody or more than one antibody.

In one respect, the present disclosure provides an antibody or antigen-binding fragment thereof which binds to GPRC5D, comprising:

In some embodiments, the antibody or antigen-binding fragment thereof comprises at least one heavy or light chain complementarity determining region (CDR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 9, 10, 11, 12, 13, 14, 17, 18, 19, 20, 21, 22, 25, 26, 27, 28, 29, 30, 33, 34, 35, 36, 37, 38, 41, 42, 43, 44, 45, 46, 49, 50, 51, 52, 53, 54, 57, 58, 59, 60, 61, 62, 65, 66, 67, 68, 69, 70, 73, 74, 75, 76, 77, 78, 97, 98, 99, 100, 101, 102, 105, 106, 107, 108, 109, 110, 113, 114, 115, 116, 117, 118, 121, 122, 123, 124, 125, 126, 129, 130, 131, 132, 133, 134, 137, 138, 139, 140, 141, 142, 145, 146, 147, 148, 149, 150, 153, 154, 155, 156, 157, 158, 169, 170, 171, 172, 173, 174, 177, 178, 179, 180, 181, 182, 185, 186, 187, 188, 189, and 190.

In some embodiments, the antibody or antigen-binding fragment thereof of the present disclosure comprises a VH region comprising one or two or three of HCDR1, HCDR2 and HCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 9, 10, 11, 17, 18, 19, 25, 26, 27, 33, 34, 35, 41, 42, 43, 49, 50, 51, 57, 58, 59, 65, 66, 67, 73, 74, 75, 97, 98, 99, 105, 106, 107, 113, 114, 115, 121, 122, 123, 129, 130, 131, 137, 138, 139, 145, 146, 147, 153, 154, 155, 169, 170, 171, 177, 178, 179, 185, 186, and 187.

In some embodiments, the antibody or antigen-binding fragment thereof of the present disclosure comprises a VL region comprising one or two or three of LCDR1, LCDR2 and LCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 5, 6, 12, 13, 14, 20, 21, 22, 28, 29, 30, 36, 37, 38, 44, 45, 46, 52, 53, 54, 60, 61, 62, 68, 69, 70, 76, 77, 78, 100, 101, 102, 108, 109, 110, 116, 117, 118, 124, 125, 126, 132, 133, 134, 140, 141, 142, 148, 149, 150, 156, 157, 158, 172, 173, 174, 180, 181, 182, 188, 189, and 190.

In some embodiments, the antibody or antigen-binding fragment thereof of the present disclosure comprises:

In some embodiments, the antibody or antigen-binding fragment thereof of the present disclosure comprises:

In some embodiments, the antibody or antigen-binding fragment thereof of the present disclosure comprises:

In some embodiments, the antibody or antigen-binding fragment thereof of the present disclosure comprises:

In some embodiments, the antibody or antigen-binding fragment thereof of the present disclosure comprises:

In some embodiments, the antibody or antigen-binding fragment thereof of the present disclosure comprises a VH region having an amino acid sequence as set forth in SEQ ID NOs: 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 103, 111, 119, 127, 135, 143, 151, 159, 175, 183, or 191, or a homologous sequence thereof having at least 80% sequence identity to SEQ ID NOs: 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 103, 111, 119, 127, 135, 143, 151, 159, 175, 183, or 191.

In some embodiments, the antibody or antigen-binding fragment thereof of the present disclosure comprises a VL region having an amino acid sequence as set forth in SEQ ID NOs: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 104, 112, 120, 128, 136, 144, 152, 160, 176, 184, or 192, or a homologous sequence thereof having at least 80% sequence identity to SEQ ID NOs: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 104, 112, 120, 128, 136, 144, 152, 160, 176, 184, or 192.

In some embodiments, the antibody or antigen-binding fragment thereof of the present disclosure comprises a VH/VL amino acid sequence pair selected from the group consisting of SEQ ID NOs: 7/8, 15/16, 23/24, 31/32, 39/40, 47/48, 55/56, 63/64, 71/72, 79/80, 103/104, 111/112, 119/120, 127/128, 135/136, 143/144, 151/152, 159/160, 175/176, 183/184, or 191/192.

In some embodiments, the antibody or antigen-binding fragment thereof of the present disclosure further comprises one or more amino acid residue substitutions or modifications yet retains binding affinity to GPRC5D. In some embodiments, at least one of the substitutions or modifications is in one or more of the CDR sequences of the VH region or VL region. In some embodiments, at least one of the substitutions or modifications is in one or more of the non-CDR sequences of the VH region or VL region. In some embodiments, the antibody or antigen-binding fragment thereof of the present disclosure further comprises one or more non-natural amino acid (NNAA) substitution. In some embodiments, the NNAA is capable of being conjugated.

In some embodiments, the antibody or antigen-binding fragment thereof of the present disclosure has one or more binding properties selected from the group consisting of:

In another aspect, the present disclosure provides an antibody or antigen-binding fragment thereof, which competes for binding to GPRC5D with the antibody or antigen-binding fragment thereof as described above.

In some embodiments, the antibody or antigen-binding fragment thereof of the present disclosure is a chimeric, a humanized or a human antibody or an antigen-binding fragment thereof.

In some embodiments, the antibody or antigen-binding fragment thereof of the present disclosure is a labeled antibody, a bivalent antibody, an anti-idiotypic antibody or a fusion protein.

In some embodiments, the antibody or antigen-binding fragment thereof of the present disclosure is a diabody, a Fab, a Fab′, a F(ab′), a Fd, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv), a bispecific dsFv (dsFv-dsFv′), a disulfide stabilized diabody (ds diabody), a single-chain antibody molecule (scFv), an scFv dimer (bivalent diabody), a camelized single domain antibody, a nanobody, a domain antibody, or a bivalent domain antibody.

In some embodiments, the antibody or antigen-binding fragment thereof of the present disclosure further comprises an Fc region. In some embodiments, the Fe region is an Fc region of human immunoglobulin (Ig). In some embodiments, the Fc region is an Fc region of human IgG. In some embodiments, the Fc region is derived from human IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 161-163.

In some embodiments, the light chain of the antibody or antigen-binding fragment thereof of the present disclosure is a λ light chain or a κ light chain.

In some embodiments, the antibody or antigen-binding fragment thereof of the present disclosure is a bispecific or multi-specific antibody or an antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof of the present disclosure is capable of specifically binding to one or more additional antigens other than GPRC5D, or a second epitope on GPRC5D. In some embodiments, the one or more additional antigens other than GPRC5D are selected from the group consisting of KRAS, ERK, XPO1, mTORC1/2, PAK4, NAMPT, ATR, EGFR, FGFR, VEGF, LILRB, c-MET, Her2, Her3, CTLA4, GITA, CD112R, CD2, CD3, CD7, CD16, CD19, CD20, CD24, CD27, CD30, CD34, CD37, CD39, CD70, CD73, CD83, CD28, CD80 (B7-1), CD86 (B7-2), CD40, CD40L (CD154), CD47, SIRPα, CD122, CD137, CD137L, OX40 (CD134), OX40L (CD252), BCMA (e.g., BCMA02), PSMA, CLDN18 (e.g., CLDN18.2), NKG2C, 4-1BB, LIGHT, PVRIG, SLAMF7, HVEM, BAFFR, ICAM-1, 2B4, LFA-1, GITR, ICOS (CD278), ICOSLG (CD275), LAG3 (CD223), A2AR, B7-H3 (CD276), B7-H4 (VTCN1), B7-H5, BTLA (CD272), BTLA, CD160, CTLA-4 (CD152), IDO1, IDO2, ILT3, TDO, KIR, LAIR-1, NOX2, PD-1, PD-L1, PD-L2, TIM-3, VISTA, SIGLEC-7 (CD328), SIGLEC-9 (CD329), SIGLEC-15, TIGIT, PVR (CD155), and TGFβ.

In some embodiments, the antibody or antigen-binding fragment thereof of the present disclosure is a bispecific antibody or antigen-binding fragment thereof that binds to GPRC5D and CD3. In some embodiments, the bispecific antibody or antigen-binding fragment thereof comprises a GPRC5D binding moiety and a CD3 binding moiety. In some embodiments, the CD3 binding moiety comprises:

In some embodiments, the CD3 binding moiety comprises:

In some embodiments, the CD3 binding moiety comprises:

In some embodiments, the CD3 binding moiety comprises a VH/VL amino acid sequence pair of SEQ ID NOs: 87/88 or 95/96.

In some embodiments, the CD3 binding moiety further comprises a constant domain CL and a constant domain CH1. In some embodiments, the constant domains CL and CH1 are replaced by each other.

In some embodiments, the bispecific antibody or multi-specific antibody or antigen-binding fragment thereof of the present disclosure further comprises an Fc region. In some embodiments, the bispecific antibody or multi-specific antibody or antigen-binding fragment thereof of the present disclosure further comprises an Fc region of human immunoglobulin (Ig). In some embodiments, the bispecific antibody or multi-specific antibody or antigen-binding fragment thereof of the present disclosure further comprises an Fc region of human IgG. In some embodiments, the Fc region is derived from human IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc region comprises one or more amino acid substitutions selected from the group consisting of: L234A, L235A, S354C, T366W, Y349C, T366S, L368A, and Y407V (according to EU numbering). In some embodiments, the Fc region comprises L234A and L235A (according to EU numbering) amino acid substitutions. In some embodiments, the Fc region of one heavy chain comprises S354C and T366W (according to EU numbering) amino acid substitutions, and the Fc region of the other heavy chain comprises Y349C, T366S, L368A, and Y407V (according to EU numbering) amino acid substitutions.

In some embodiments, the antibody or antigen-binding fragment thereof of the present disclosure is linked to one or more conjugate moieties. In some embodiments, the conjugate moiety comprises a clearance-modifying agent, a chemotherapeutic agent, a toxin, a radioactive isotope, a lanthanide, a detectable label, a DNA-alkylator, a topoisomerase inhibitor, a tubulin-binder, a purification moiety or other anticancer drugs. In some embodiments, the conjugate moiety is covalently attached either directly or via a linker.

In another aspect, the present disclosure provides a chimeric antigen receptor comprising the antibody or antigen-binding fragment thereof provided herein, a transmembrane region and an intracellular signal region. In some embodiments, the transmembrane region comprises a transmembrane region of CD3, CD4, CD8 or CD28. In some embodiments, the intracellular signal region is selected from the group consisting of: an intracellular signal regions sequence of CD3 (e.g. CD3ζ), FcγR1, CD27, CD28, CD137, CD134, MyD88, CD40, CD278, TLRs, or a combination thereof. In some embodiments, the antigen-binding fragment of the chimeric antigen receptor is a scFv. In some embodiments, the chimeric antigen receptor is grafted onto an allogeneic cell, an autologous cell or a xenogeneic cell. In some embodiments, the chimeric antigen receptor is grafted onto an immune effector cell. In some embodiments, the chimeric antigen receptor is grafted onto a T cell, a natural killer cell, a macrophage cell, or a tumor-infiltrating lymphocyte.

In another aspect, the present disclosure provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, and/or the chimeric antigen receptor of the present disclosure, and one or more pharmaceutically acceptable carriers.

In another aspect, the present disclosure provides an isolated polynucleotide encoding the antibody or antigen-binding fragment thereof and/or the chimeric antigen receptor of the present disclosure.

In another aspect, the present disclosure provides a vector comprising the isolated polynucleotide of the present disclosure.

In another aspect, the present disclosure provides a host expression system comprising the vector of the present disclosure or having the polynucleotide of the present disclosure integrated into genome thereof. In some embodiments, the host expression system of the present disclosure is a microorganism, a yeast, or a mammalian cell. In some embodiments, the microorganism is selected from the group consisting ofand. In some embodiments, the yeast is. In some embodiments, the mammalian cell is selected from the group consisting of COS, CHO-S, CHO-KI, HEK-293, and 3T3 cells.

In another aspect, the present disclosure provides a virus comprising the vector of the present disclosure.

In another aspect, the present disclosure provides a kit comprising the antibody or antigen-binding fragment thereof of the present disclosure and/or the chimeric antigen receptor of the present disclosure and/or the pharmaceutical composition of the present disclosure, and a second therapeutic agent.

In another aspect, the present disclosure provides a method of expressing the antibody or antigen-binding fragment thereof of the present disclosure and/or the chimeric antigen receptor of the present disclosure, comprising culturing the host expression system of the present disclosure under the condition at which the antibody or antigen-binding fragment of the present disclosure is expressed.

In another aspect, the present disclosure provides a method of treating, preventing or alleviating a disease, disorder or condition in a subject, comprising administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment thereof and/or the chimeric antigen receptor and/or the pharmaceutical composition of the present disclosure.

In another aspect, the present disclosure provides use of the antibody or antigen-binding fragment thereof and/or the chimeric antigen receptor and/or the pharmaceutical composition of the present disclosure in the manufacture of a medicament for treating a GPRC5D-related disease, disorder or condition in a subject.

In another aspect, the present disclosure provides use of the antibody or antigen-binding fragment thereof and/or the chimeric antigen receptor and/or the pharmaceutical composition of the present disclosure in the manufacture of a diagnostic reagent for diagnosing a GPRC5D-related disease, disorder or condition.

In some embodiments, the disease, disorder or condition is cancer. In some embodiments, the cancer is a solid tumor or hematologic tumor. In some embodiments, the disease, disorder or condition is a GPRC5D-expressing B cell cancer. In some embodiments, the disease, disorder or condition is selected from the group consisting of lung cancer (e.g., non-small-cell lung cancer (NSCLC), small cell lung cancer (SCLC), adenocarcinoma of the lung, or squamous cell carcinoma of the lung), peritoneal cancer, carcinoid cancer, bone cancer, pancreatic cancer, primitive neuroectodermal tumor, skin cancer, gallbladder cancer, cancer of the head or neck, squamous cell cancer, uterine cancer, ovarian cancer, rectal cancer, prostate cancer, bladder cancer (e.g., urothelial cancer), cancer of the anal region (e.g., anal squamous cell carcinoma), gastric or stomach cancer (e.g., gastrointestinal cancer), esophageal cancer, colon cancer, breast cancer, uterine cancer, liver cancer (e.g., hepatoblastoma, hepatocellular carcinoma/hepatoma, or hepatic carcinoma), cholangiocarcinoma, sarcoma, colorectal cancer, carcinoma of the fallopian tubes, salivary gland carcinoma, carcinoma of the cervix, endometrial or uterine carcinoma, osteosarcoma, carcinoma of the vagina, carcinoma of the vulva, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, cancer of the nasopharynx, sarcoma of soft tissue, polycythemia vera, cancer of the urethra, cancer of the penis, cancer of the kidney or ureter (e.g., rhabdoid tumor of the kidney), cutaneous T-cell lymphoma, medulloblastoma, nephroblastoma, myelodysplastic syndrome, chronic and non-chronic myeloproliferative disorder, choroid plexus papilloma, renal cell carcinoma, carcinoma of the renal pelvis, neoplasms of the central nervous system (CNS), soft tissue sarcoma (e.g., rhabdomyosarcoma, fibrosarcoma, Kaposi's sarcoma), spinal axis tumors, glioma (e.g., ependymoma, astrocytoma, anaplastic astrocytoma, oligodendroglioma, eye cancer (e.g., retinoblastoma), brain stem glioma, or mixed glioma such as oligoastrocytoma), brain tumor (e.g., glioblastoma/glioblastoma multiforme (GBM), non-glioblastoma brain tumor, or meningioma), cutaneous or intraocular melanoma, thrombocythemia, mesothelioma, mycosis fungoides, Sezary syndrome, idiopathic myelofibrosis, solitary plasmacytoma, vestibular schwannoma, Ewing's sarcoma, chondrosarcoma, MYH associated polyposis, pituitary adenoma, pediatric cancers such as pediatric sarcomas (e.g., neuroblastoma, rhabdomyosarcoma, and osteosarcoma), hematological cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia (e.g., lymphocytic/lymphoblastic leukemia), chronic or acute leukemia, mast cell leukemia, lymphocytic lymphomas, primary CNS lymphoma, chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), chronic myeloid leukemia (CML), acute myeloid leukemia (AML), chronic myelomonocytic leukemia (CMML), chronic lymphoblastic leukemia, acute lymphoblastic leukemia, hairy cell leukemia (HCL), Burkitt's lymphoma (BL), multiple myeloma (e.g., relapsed or refractory multiple myeloma), T or B cell lymphoma, mantle cell lymphoma (MCL) (e.g., relapsed or refractory mantle cell lymphoma), malignant melanoma, diffuse large B cell lymphoma (DLBCL), DLBCL that results from follicular lymphoma, high-grade B-cell lymphoma, primary mediastinal large B-cell lymphoma, follicular lymphoma (FL), and primary mediastinal B-cell lymphoma. In some embodiments, the disease, disorder or condition is multiple mycloma.

In some embodiments, the subject is human.

In some embodiments, the administration is through a parenteral route comprising subcutaneous, intraperitoneal, intravenous, intramuscular, or intradermal injection; or a non-parenteral route comprising transdermal, oral, intranasal, intraocular, sublingual, rectal, or topical.

In some embodiments, the method of treating, preventing or alleviating a disease, disorder or condition in a subject further includes administering to the subject in need thereof an additional therapeutic agent. In some embodiments, the additional therapeutic agent is selected from the group consisting of: an active agent, an imaging agent, a cytotoxic agent, and angiogenesis inhibitor, a kinase inhibitor, a co-stimulation molecule agonist, a co-inhibition molecule blocker, an adhesion molecule blocker, an anti-cytokine antibody or functional fragment thereof, a detectable label or reporter, an antimicrobial, a gene editing agent, a beta agonist, an viral RNA inhibitor, a polymerase inhibitor, an interferon, and a microRNA. In some embodiments, the additional therapeutic agent is administered to the subject in need before, after or simultaneously with the antibody or antigen-binding fragment thereof and/or the pharmaceutical composition of the present disclosure.

In another aspect, the present disclosure provides a method of inactivating a GPRC5D-expressing cell in vivo or in vitro, comprising contacting the GPRC5D-expressing cell with the antibody or antigen-binding fragment thereof and/or the chimeric antigen receptor and/or the pharmaceutical composition of the present disclosure.

In another aspect, the present disclosure provides a method of modulating GPRC5D activity in a GPRC5D-expressing cell, comprising exposing the GPRC5D-expressing cell to the antibody or antigen-binding fragment thereof and/or the chimeric antigen receptor and/or the pharmaceutical composition of the present disclosure.